US2026053882A1PendingUtilityA1

Logic gated protein actuators

60
Assignee: UNIV PRINCETONPriority: Aug 23, 2022Filed: Aug 23, 2023Published: Feb 26, 2026
Est. expiryAug 23, 2042(~16.1 yrs left)· nominal 20-yr term from priority
Inventors:MUIR THOMAS WKOFOED CHRISTIANLEE GIHOONGRAMESPACHER JOSEFTAY NICHOLASYE XUANJIAERKALO GIRUM
C12N 9/00C07K 2319/70C07K 16/40C07K 16/2809C07K 14/71A61K 49/0002A61P 35/00H10K 19/901H10K 10/701C07K 14/4702A61K 38/00C12N 15/62
60
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Claims

Abstract

A logic-gated protein device in which proximity-gated protein trans-splicing governs formation of an active protein from two otherwise inactive fragments.

Claims

exact text as granted — not AI-modified
1 . A logic-gated protein actuator system, comprising:
 a mixture comprising   a first component comprising a first caged split intein fragment fused to a first protein-of-interest fragment and fused to a first antigen-binding domain configured to bind to a first surface antigen; and   a second component comprising a second caged split intein fragment fused to a second protein-of-interest fragment and fused to a second antigen-binding domain configured to bind to a second surface antigen,   wherein the first caged split intein fragment and the second caged split intein fragment are capable of undergoing protein trans-splicing to form a protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment when the first antigen-binding domain and the second antigen-binding domain are proximal, and   wherein the first caged split intein fragment and the second caged split intein fragment are an actuator.   
     
     
         2 . The logic-gated protein actuator system of  claim 1 ,
 wherein the first surface antigen and the second surface antigen are different,   wherein the mixture does not comprise a third component comprising the first caged split intein fragment fused to the first protein-of-interest fragment and fused to the second antigen-binding domain,   wherein the mixture does not comprise a fourth component comprising the second caged split intein fragment fused to the second protein-of-interest fragment and fused to the first antigen-binding domain, and   wherein the logic-gated protein actuator system is a first-surface-antigen-AND-second-surface-antigen logic gate.   
     
     
         3 . The logic-gated protein actuator system of  claim 1 ,
 wherein the mixture comprises a fifth component comprising the second caged split intcin fragment fused to the second protein-of-interest fragment and fused to a third antigen-binding domain configured to bind to a third surface antigen,   wherein the third surface antigen is different from the first surface antigen and from the second surface antigen,   wherein the first caged split intein fragment and the second caged split intein fragment are capable of undergoing protein trans-splicing to form the protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment when the third antigen-binding domain and the first antigen-binding domain are proximal,   wherein the logic-gated protein actuator system is a first-surface-antigen-AND-either-second-surface-antigen-OR-third-surface-antigen logic gate.   
     
     
         4 . The logic-gated protein actuator system of  claim 3 ,
 wherein the mixture comprises at least one additional component,   wherein each additional component comprises the second caged split intein fragment fused to the second protein-of-interest fragment and fused to an additional antigen-binding domain configured to bind to an additional surface antigen,   wherein each additional surface antigen of each additional component is different from the first surface antigen, the second surface antigen, the third surface antigen, and each other additional surface antigen,   wherein the first caged split intein fragment and the second caged split intein fragment are capable of undergoing protein trans-splicing to form the protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment when the additional antigen-binding domain is proximal to the third antigen-binding domain, the second antigen-binding domain, the first antigen-binding domain, or any other additional binding domain,   wherein the logic-gated protein actuator system is a first-surface-antigen-AND-either-second-surface-antigen-OR-third-surface-antigen-OR-additional-surface-antigen(s) logic gate.   
     
     
         5 . The logic-gated protein actuator system of  claim 1 ,
 wherein the mixture comprises a fifth component comprising the second caged split intein fragment fused to a modified second protein-of-interest fragment and fused to a third antigen-binding domain configured to bind to a third surface antigen,   wherein the third surface antigen is different from the first surface antigen and from the second surface antigen,   wherein the first caged split intein fragment and the second caged split intein fragment of the fifth component are capable of undergoing protein trans-splicing to form a defective protein of interest from the first protein-of-interest fragment and the modified second protein-of-interest fragment when the third antigen-binding domain and the first antigen-binding domain are proximal,   wherein the defective protein of interest is different from the protein of interest,   wherein the logic-gated protein actuator system is a first-surface-antigen-AND-second-surface-antigen-NOT-third-surface-antigen logic gate.   
     
     
         6 . The logic-gated protein actuator system of  claim 5 ,
 wherein the mixture comprises a sixth component comprising the second caged split intein fragment fused to the modified second protein-of-interest fragment and fused to a fourth antigen-binding domain configured to bind to a fourth surface antigen,   wherein the fourth surface antigen is different from the first surface antigen, from the second surface antigen, and from the third surface antigen,   wherein the first caged split intein fragment and the second caged split intein fragment of the sixth component are capable of undergoing protein trans-splicing to form the defective protein of interest from the first protein-of-interest fragment and the modified second protein-of-interest fragment when the fourth antigen-binding domain and the first antigen-binding domain are proximal,   wherein the logic-gated protein actuator system is a first-surface-antigen-AND-second-surface-antigen-NOT-third-surface-antigen-NOT-fourth-surface-antigen logic gate.   
     
     
         7 . The logic-gated protein actuator system of  claim 6 ,
 wherein the mixture comprises at least one additional component comprising the second caged split intcin fragment fused to the modified second protein-of-interest fragment and fused to an additional antigen-binding domain configured to bind to an additional surface antigen,   wherein the additional surface antigen is different from the first surface antigen, from the second surface antigen, from the third surface antigen, and from the fourth surface antigen,   wherein the first caged split intein fragment and the second caged split intein fragment of the additional component are capable of undergoing protein trans-splicing to form the defective protein of interest from the first protein-of-interest fragment and the modified second protein-of-interest fragment when the additional antigen-binding domain and the first antigen-binding domain are proximal,   wherein the logic-gated protein actuator system is a first-surface-antigen-AND-second-surface-antigen-NOT-third-surface-antigen-NOT-fourth-surface-antigen-NOT-additional-surface-antigen logic gate.   
     
     
         8 . The logic-gated protein actuator system of  claim 5 ,
 wherein the first surface antigen and the second surface antigen are identical and   wherein the logic-gated protein actuator system is a first-surface-antigen-NOT-third-surface-antigen logic gate.   
     
     
         9 . The logic-gated protein actuator system of  claim 6 ,
 wherein the first surface antigen and the second surface antigen are identical and   wherein the logic-gated protein actuator system is a first-surface-antigen-AND-[third-surface-antigen-NOR-fourth-surface-antigen] logic gate.   
     
     
         10 . The logic-gated protein actuator system of  claim 1 ,
 wherein the first surface antigen and the second surface antigen are different,   wherein the mixture comprises a third component comprising the first caged split intein fragment fused to the first protein-of-interest fragment and fused to the second antigen-binding domain,   wherein the mixture comprises a fourth component comprising the second caged split intcin fragment fused to the second protein-of-interest fragment and fused to the first antigen-binding domain,   wherein the first caged split intein fragment and the second caged split intein fragment are capable of undergoing protein trans-splicing to reconstitute the protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment when two first antigen-binding domains are proximal,   wherein the first caged split intein fragment and the second caged split intein fragment are capable of undergoing protein trans-splicing to reconstitute the protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment when two second antigen-binding domains are proximal, and   wherein the logic-gated protein actuator system is a first-surface-antigen-OR-second-surface-antigen logic gate.   
     
     
         11 . The logic-gated protein actuator system of  claim 10 ,
 wherein the mixture comprises a fifth component comprising the first caged split intein fragment fused to the first protein-of-interest fragment and fused to a third antigen-binding domain,   wherein the mixture comprises a sixth component comprising the second caged split intein fragment fused to the second protein-of-interest fragment and fused to a fourth antigen-binding domain,   wherein the third antigen-binding domain is different from the second antigen-binding domain and from the first antigen-binding domain,   wherein the first caged split intein fragment and the second caged split intein fragment are capable of undergoing protein trans-splicing to reconstitute the protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment when the third antigen-binding domain is proximal to the first antigen-binding domain or the second antigen-binding domain or two third antigen-binding domains are proximal . . . and   wherein the logic-gated protein actuator system is a first-surface-antigen-OR-second-surface-antigen-OR-third-surface-antigen logic gate.   
     
     
         12 . The logic-gated protein actuator system of  claim 11 ,
 wherein the mixture comprises at least one additional pair of components,   wherein the first part of the additional pair of components comprises the first caged split intein fragment fused to the first protein-of-interest fragment and fused to an additional antigen-binding domain configured to bind to an additional surface antigen,   wherein the second part of the additional pair of components comprises the second caged split intein fragment fused to the second protein-of-interest fragment and fused to the additional antigen-binding domain,   wherein each additional surface antigen is different from the first surface antigen, the second surface antigen, the third surface antigen, and each other additional surface antigen,   wherein the first caged split intein fragment and the second caged split intein fragment are capable of undergoing protein trans-splicing to form the protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment when the additional antigen-binding domain is proximal to the third antigen-binding domain, the second antigen-binding domain, the first antigen-binding domain, or any additional binding domain, and   wherein the logic-gated protein actuator system is a first-surface-antigen-OR-second-surface-antigen-OR-third-surface-antigen-OR-additional-surface-antigen(s) logic gate.   
     
     
         13 . The logic-gated protein actuator system of  claim 1 ,
 wherein the first caged split intein fragment is eNrdJ-1N cage -K114AK116A and   wherein the second caged split intein fragment is eNrdJ-1C cage .   
     
     
         14 . The logic-gated protein actuator system of  claim 1 ,
 wherein the first caged split intein fragment is eNrdJ-1C cage  and   wherein the second caged split intein fragment is eNrdJ-1N cage -K114AK116A.   
     
     
         15 . The logic-gated protein actuator system of  claim 1 ,
 wherein the pair of the first caged split intein fragment and the second caged split intein fragment are selected from the group of pairs consisting of eNrdJ-1N cage  & eNrdJ-1C cage , NrdJ-IN cage  & NrdJ-1C cage , NpUN cage  & NpuC cage , Gp41-1N cage  & Gp41-1C cage , Gp41-8N cage  & Gp41-80 cage , eNrdJ-1C cage  & eNrdJ-1N cage , NrdJ-1C cage  & NrdJ-1N cage , NpuC cage  & NpuN cage , Gp41-1C cage  & Gp41-1N cage , and Gp41-8C cage  & Gp41-8N cage .   
     
     
         16 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest comprises a toxin.   
     
     
         17 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest comprises an imaging agent.   
     
     
         18 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest comprises an antibody epitope.   
     
     
         19 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest comprises an antigen or antigen fragment to which an antibody or an antibody paratope or a T-cell or a T-cell receptor can bind or a protein, protein fragment, antigen, antibody, or antigen-antibody complex to which a complement protein can bind.   
     
     
         20 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest comprises an enzyme.   
     
     
         21 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest is capable of binding a toxin.   
     
     
         22 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest is capable of binding an imaging agent.   
     
     
         23 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest is capable of binding an antibody epitope.   
     
     
         24 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest is capable of binding an antigen or an antigen fragment to which an antibody or an antibody paratope or a T-cell or a T-cell receptor can bind or a protein, protein fragment, antigen, antibody, or antigen-antibody complex to which a complement protein can bind.   
     
     
         25 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest is capable of binding an enzyme.   
     
     
         26 . The logic-gated protein actuator system of  claim 1 ,
 wherein the protein of interest is SpyCatcher003.   
     
     
         27 . The logic-gated protein actuator system of  claim 1 ,
 wherein the first protein of interest fragment is SpyN 1-73  and   wherein the second protein of interest fragment is SpyC 74-113.      
     
     
         28 . The logic-gated protein actuator system of  claim 1 ,
 wherein the first protein of interest fragment is SpyC 74-113  and   wherein the second protein of interest fragment is SpyN 1-73.      
     
     
         29 . The logic-gated protein actuator system of  claim 1 ,
 wherein the pair of the first protein-of-interest fragment and the second protein-of-interest fragment are selected from the group of pairs consisting of SpyN 1-24  & SpyC 25-113 , SpyN 1-42  & SpyC 43-113 , SpyN 1-55  & SpyC 56-113 , SpyN 1-82  & SpyC 83-113 , SpyN 1-90  & SpyC 91-113 , SpyC 25-113  & SpyN 1-24 , SpyC 43-113  & SpyN 1-42 , SpyC 56-113  & SpyN 1-55 , SpyC 83-113  & SpyN 1-82 , and SpyC 91-113  & SpyN 1-90 .   
     
     
         30 . The logic-gated protein actuator system of  claim 1 ,
 wherein the mixture further comprises a SpyTag003-biotin conjugate.   
     
     
         31 . The logic-gated protein actuator system of  claim 1 ,
 wherein the mixture further comprises a SpyTag003-biotin conjugate and a Streptavidin-Saporin conjugate.   
     
     
         32 . The logic-gated protein actuator system of  claim 1 ,
 wherein the mixture further comprises a SpyTag003-AF594 conjugate.   
     
     
         33 . The logic-gated protein actuator system of  claim 1 ,
 wherein the mixture further comprises a SpyTag003-APEX2 conjugate.   
     
     
         34 . The logic-gated protein actuator system of  claim 1 ,
 wherein the first surface antigen is selected from the group consisting of HER2, EGFR, and EpCAM and   wherein the second surface antigen is selected from the group consisting of HER2, EGFR, and EpCAM.   
     
     
         35 . The logic-gated protein actuator system of  claim 3 ,
 wherein the first surface antigen is selected from the group consisting of HER2, EGFR, and EpCAM and   wherein the second surface antigen is selected from the group consisting of HER2, EGFR, and EpCAM,   wherein the third surface antigen is selected from the group consisting of HER2, EGFR, and EpCAM.   
     
     
         36 . The logic-gated protein actuator system of  claim 4 ,
 wherein each surface antigen is selected from the group consisting of HER2, EGFR, and EpCAM.   
     
     
         37 . A method of conditionally forming a protein of interest, comprising:
 providing a mixture comprising
 a first component comprising a first caged split intein fragment fused to a first protein-of-interest fragment and fused to a first antigen-binding domain configured to bind to a first surface antigen; and 
 a second component comprising a second caged split intein fragment fused to a second protein-of-interest fragment and fused to a second antigen-binding domain configured to bind to a second surface antigen, 
   bringing the mixture into contact with a cell having a surface, and   when the first surface antigen and the second surface antigen are proximal on the surface of the cell, allowing the first antigen-binding domain to bind to the first surface antigen and the second antigen-binding domain to bind to the second surface antigen, and allowing the first caged split intein fragment and the second caged split intein fragment to undergo protein trans-splicing to form a protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment,   wherein the first caged split intein fragment and the second caged split intein fragment are an actuator.   
     
     
         38 . The method of  claim 37 , further comprising
 allowing the protein of interest to bind a toxin, and   allowing the toxin to damage or kill the cell.   
     
     
         39 . Treating a cancer by the method of  claim 38 ,
 further comprising administering the mixture and the toxin to a patient,   wherein the cell is a cancerous cell.   
     
     
         40 . The method of  claim 37 , further comprising
 allowing the protein of interest to bind an imaging agent, and   using the imaging agent to image the cell.   
     
     
         41 . Diagnosing a hyperproliferative disorder by the method of  claim 40 ,
 further comprising administering the mixture and the imaging agent to a patient,   wherein the cell is a hyperproliferative cell.   
     
     
         42 . The method of  claim 37 ,
 wherein the protein of interest comprises a toxin and   further comprising allowing the toxin to damage or kill the cell.   
     
     
         43 . The method of  claim 37 ,
 wherein the protein of interest comprises an imaging agent and   further comprising using the imaging agent to image the cell.

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